1. Field of the Invention
The present invention relates to antagonists of RF-amide neuropeptides and to methods of using same to attenuate the effects of drug addiction, drug tolerance, drug dependence or of abstinence syndrome, and to treat certain other conditions.
2. Description of the Prior Art
Opiate tolerance, dependence, and abuse represent major medical and social problems. Neuropeptide FF (or NPFF) [Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 or F8F-amide] 1, together with the related mammalian neuropeptides NPAF and the N-terminally extended (Ser-Gln-Ala-) have been identified as high-affinity endogenous ligands for a novel neuropeptide Y-like human orphan G-protein coupled receptor HLWAR77 [Elshourbagy, et al., J. Biol. Chem. 2000, 275, 25965-25971]. Receptors activated by 1 have also recently been isolated from human and rat central nervous system (CNS) tissue [Bonini, et al., J. Biol. Chem. 2000, 275, 39324-39331]. The octapeptide NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 was originally isolated by Yang et al. from bovine brain (Yang et al., Proc. Natl. Acad. Sci., 1985, 82, 7757-61). It has also been referred to as xe2x80x9cmorphine-modulating peptidexe2x80x9d or xe2x80x9cFMRFa-like mammalian octapeptidexe2x80x9d (Panula et al., Med. Biology, 1987, 65, 127-35 or Neuropeptide FF (Kivipelto et al., Journal of Comparative Neurology). There are reasons to suspect that NPFF may be an xe2x80x9canti-opiate peptidexe2x80x9d: NPFF is localized in several brain regions rich in endogenous opioids (Ferrarese et al., Regulatory Peptides, 1986, 13, 245-52; Panula et al., Med. Biology, 65:127-35 (1987)), is released from the brain by morphine infusion (Tang et al., Proc. Natl. Acad. Sci., 1984, 81, 5002-5), and potently antagonizes analgesic effects of morphine and certain endogenous opioid peptides (Tang et al., Proc. Natl. Acad. Sci., 1984, 81, 5002-5; Yang et al., Proc. Natl. Acad. Sci., 1985, 82, 7757-7761; Yang et al., Prog. Clin. Biol. Res., 1985, 192, 313-22). IgG from NPFF antiserum augments morphine and stress-induced analgesia (Kavaliers et al., Peptides, 1989, 10, 741-5).
There is also evidence that NPFF may participate in opiate tolerance and dependence. IgG prepared from FMRFa antiserum cross-reacts with NPFF and interferes with morphine tolerance (Tang et al., Proc. Natl. Acad. Sci., 1984, 81, 5002-5). NPFF levels in CSF are markedly increased in opiate dependent rats as compared with non-dependent rats (Malin et al., Peptides, 1990, 11, 969-972). NPFF (2 xcexcg i.c.v.) precipitates opiate abstinence syndrome in morphine-dependent rats (Malin et al., Peptides, 1990, 11, 277-280), and NPFF (15 xcexcg i.c.v.) induces a quasi-morphine-abstinence syndrome (QMAS) in opiate-naive rats (Malin et al., Peptides, 1990, 11, 277-280) (see also Guzman et al., Neuropeptides 1989, 14, 253-261; Majane et al., Peptides, 1987, 8, 657-662; Majane et al., Peptides, 1988, 9, 1137-1144). Third ventricle infusion of IgG from NPFF antiserum reverses opiate dependence, as evidenced by prevention of naloxone-precipitated abstinence syndrome in morphine-dependent rats (Majane et al., Peptides, 1990, 11, 969-972). The mechanism of action of NPFF is not understood as yet, but a recent receptor binding study in spinal cord membranes suggested that the neuropeptide binds to specific NPFF receptors. The 125I-Y8Fa binding site showed high affinity for NPFF, whereas opioid ligands failed to compete for binding (Allard et al., Brain Res. 1989, 500, 169-176).
Neuropeptide FF has been implicated in pain modulation, morphine tolerance, and morphine abstinence [Rothman, Synapse 1992, 12, 129-138]. Intracerebroventricular (icv) pretreatment with immunoglobulin G (IgG) from antiserum of 1 restored the analgesic effect of icv morphine in morphine-tolerant rats [Lake et al., Neurosci. Lett. 1991, 132, 29-32] and potentiated the anti-opioid effect of 1 [Kavaliers et al., Peptides 1991, 12, 235-239].
It has been recognized that an NPFF antagonist would be useful as a probe for determining the physiological role of endogenous NPFF, as well as further ascertaining its role in opiate dependence, tolerance and abstinence. Centrally administered neuropeptide FF also has been known to precipitate quasi-morphine abstinence syndrome (QMAS) in opiate-naive animals. Therefore, antagonists of 1 (besides their importance as pharmacological agents helpful in defining the physiological/pharmacological role of the endogenous neuropeptide) could also allow for the management of withdrawal symptoms that adversely affect the treatment of opiate abuse. Accordingly, NPFF analogs have been synthesized. One analog differs in two respects from the NPFF sequence. First, in order to reduce receptor activation, the C-terminal Arg-Phe-amide was replaced by Arg-amide. Secondly, in order to increase resistance to aminopeptidase, the N-terminal was blocked with desaminotyrosine (daY). With both N-and C-termini blocked, this peptide has increased enzyme resistance and receptor availability. DaY increases peptide binding affinity at molluscan FMRFa receptors and therefore it was used as the N-terminal block (see Payza, Peptides, 1987, 8, 1065-1074).
Desaminotyrosyl-Phe-Leu-Phe-Gln-Pro-Gln-Arg-NH2 (2), one of the first putative antagonists of 1 discovered, has indeed attenuated abstinence-like signs induced by 1 in opiate-naive rats and upon naloxone challenge in morphine-dependent animals after icv administration [Malin, et al., Peptides 1991, 12, 1011-4]. To date, 2 showed the highest potency upon icv administration in blunting behavioral effects precipitated by 1. However, this peptide analogue did not show any CNS bioavailability after systemic administration and, thus, could not be considered a potential therapeutically valuable compound.
Derivatization with 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl) at the secondary NH group of the N-terminal proline residue of the tripeptide Pro-Gln-Arg-NH2, obtained from the sequence (residues 5-7) of 1, has afforded an antagonist with significant lipid solubility to cross the blood-brain barrier (BBB) [Malin, et al., Drug Alcohol Depend. 1995, 40, 37-42; Prokai, et al., Rapid Commun. Mass Spectrom. 2000, 14, 2414-2418]. Dansyl-Pro-Gln-Arg-NH2 (3): 
dose-dependently antagonized QMAS induced by 1, and it also blunted naloxone-precipitated withdrawal symptoms in morphine-dependent rats when administered subcutaneously. In the meantime, 3 was also expected to improve resistance compared to 2 against proteolytic enzymes. Considering competitive binding against a radioiodinated analogue of 1 in a CNS membrane preparation, the micromolar range inhibition constant (Ki) of 3 has remained the sole xe2x80x9cbenchmarkxe2x80x9d measuring antagonism of the endogenous octapeptide at the receptor level. Although a recent study has identified Pro-Phe-Arg(Tic)-NH2 (3a Tic=L-1,2,3,4-tetrahydroisoquinoline-3-carboxyl) as a putative antagonist of 1 that attenuated naloxone-precipitated withdrawal symptoms in morphine-dependent rats after systemic administration [Tan, et al., Peptides 1999, 20, 1211-1217], the potency (or efficacy) of this compound was less than that of 3 in the pharmacological tests employed.
The affinity of compound 3 to the receptor labeled by the radioiodinated analogue of 1(4, [125I]-YLFQPQRF-NH2 or [125I]Y8F-amide) was confirmed using the assay adapted, and the measured Ki of 13.6+2.5 xcexcM was in good agreement with the value reported before. On the other hand, Ki=840xc2x1180 nM that was obtained for 2, showing the highest efficacy upon icv administration in blunting opiate abstinence in animals. These observations demonstrated a correlation between the binding affinity to the receptors labeled by 4 and the desired pharmacological effect as an antagonist of 1.
It is an object of the invention to provide compounds that are useful in acting as antagonists in blocking the effects of NPFF and other endogenous neuropeptides with Arg-Phe-NH2 in their C-termini, and in acting to block dependence on drugs of abuse or addiction and their subsequent abstinence syndromes.
It is a further object of the invention to provide a method by which the inventive compound/antagonist can be employed to either enhance the efficacy of morphine treatment or to prevent drug dependence and to ameliorate the effects of abstinence syndromes.
The above and other objects are realized by the invention, one embodiment of which relates to a compound having the formula I below: 
where
R1=H, C1-C6 alkyl, cycloalkyl, 
R2=H, C1-C6 alkyl, cycloalkyl
W=CnH2n-mxe2x80x94NH (n=1-6, m=0, 2, or 4), 
Z=CONR8(CH2)n, CONR8(CH2)nCO, P(CH3)OCHR8OCOR9, SO2, SO2(CH2)n, SO2(CH2)nCO, SO2NR8(CH2)n, SO2NR8(CH2)nCO, n=1-4
R4=H, (CH2)nOH, (CH2)nOCOR10, (CH2)nNR10R11, (CH2)nCONR10R11, n=0-4
R5=H, (CH2)nNR12R13, n=0-4
R6=H, (CH2)nNR14R15, n=0-4
R7=H, C1-C6 alkyl, cycloalkyl; R8=H, C1-C6 alkyl, cycloalkyl; R9=H, C1-C6 alkyl, cycloalkyl;
R10=H, C1-C6 alkyl, cycloalkyl; R11=H, C1-C6 alkyl, cycloalkyl; R12=H, C1-C6 alkyl, cycloalkyl;
R13=H, C1-C6 alkyl, cycloalkyl; R14=H, C1-C6 alkyl, cycloalkyl; R15=H, C1-C6 alkyl, cycloalkyl
Dashed lines: optional; conformational constraint by (CH2)n, n=1-3, Rxe2x80x2=H or O(xe2x95x90)
An additional embodiment of the invention concerns a pharmaceutical composition for attenuating the effects of an opiate addiction, opiate dependence, opiate tolerance, opiate related abstinence syndrome, nicotine addiction, obesity, comprising at least one of the above described compounds in an amount sufficient to effect said attenuation, together with a pharmaceutically acceptable carrier.
Another embodiment of the invention comprises a method of treating an opiate addiction, opiate dependence, opiate tolerance, opiate related abstinence syndrome, nicotine addiction, obesity, comprising administering to a mammal in need of such treatment an amount of the above described peptide sufficient to effect the treatment.